Enrichment of SARS-CoV-2 sequence from nasopharyngeal swabs whilst identifying the nasal microbiome

Simultaneously characterising the genomic information of coronaviruses and the underlying nasal microbiome from a single clinical sample would help characterise infection and disease. Metatranscriptomic approaches can be used to sequence SARS-CoV-2 (and other coronaviruses) and identify mRNAs associated with active transcription in the nasal microbiome. However, given the large sequence background, unenriched metatranscriptomic approaches often do not sequence SARS-CoV-2 to sufficient read and coverage depth to obtain a consensus genome, especially with moderate and low viral loads from clinical samples. In this study, various enrichment methods were assessed to detect SARS-CoV-2, identify lineages and define the nasal microbiome. The methods were underpinned by Oxford Nanopore long-read sequencing and variations of sequence independent single primer amplification (SISPA). The utility of the method(s) was also validated on samples from patients infected seasonal coronaviruses. The feasibility of profiling the nasal microbiome using these enrichment methods was explored. The findings shed light on the performance of different enrichment strategies and their applicability in characterising the composition of the nasal microbiome

Evaluation of Arterial Histopathology and microRNA Expression That Underlie Ultrasonography Findings in Temporal Arteries of Patients with Giant Cell Arteritis

The aim of this study was to assess the interrelation between vascular ultrasonography (US) findings, histopathological data, and the expression of selected dysregulated microRNAs (miRNAs) in giant cell arteritis (GCA). The study included data on the clinical parameters, US measurements, and temporal artery biopsies (TABs) of 46 treatment-naïve patients diagnosed with GCA and 22 age-matched non-GCA patient controls. We performed a comprehensive comparative and correlation analysis along with generation of receiver operating characteristic (ROC) curves to ascertain the diagnostic performance of US examination parameters and selected miRNAs for GCA diagnosis. We showed significant differences in the US-measured intima–media thickness of the temporal arteries, the presence of a halo sign, and the presence of luminal stenosis between GCA-positive/TAB-positive, GCA-positive/TAB-negative, and non-GCA patients. Correlation analysis revealed significant associations between several histopathological parameters, US-measured intima–media thickness, and the halo sign. We found that the significant overexpression of miR-146b-5p, miR-155-5p, miR-511-5p, and miR-21-5p, and the under-expression of the miR-143/145 cluster, miR-30a-5p, and miR-125a-5p, coincides and is associated with the presence of a halo sign in patients with GCA. Notably, we determined a high diagnostic performance of miR-146b-5p, miR-21-3p, and miR-21-5p expression profiles in discriminating GCA patients from non-GCA controls, suggesting their potential utilization as putative biomarkers of GCA. Taken together, our study provides an insight into the US-based diagnostic evaluation of GCA by revealing the complex interrelation of clearly defined image findings with underlying vascular immunopathology and altered arterial tissue-specific miRNA profiles

​Histopathological changes of temporal arteries in relation to the altered expression of microRNA, clinical manifestations and ultrasound examination in the giant cell arteritis

Uvod: Poglavitni namen doktorskega dela je bil pridobiti dodaten vpogled v patogenetske mehanizme gigantoceličnega arteritisa (GCA) in povezati le-te z ultrazvočno preiskavo arterij (danes preiskavo izbora v diagnostiki gigantoceličnega vaskulitisa). V raziskavah, opravljenih v okviru doktorskega dela, smo preverili izražanje mikro RNA (miRNA) v temporalnih arterijah (TA) bolnikov in njihove genske tarče. Fenotipizirali in kvantitativno smo ocenili imunske celice, ki sestavljajo vnetni infiltrat v biopsijah temporalnih arterij (TAB) bolnikov z GCA, ter pridobljene histopatološke podatke povezali z izražanjem miRNA. Poleg tega smo ocenili medsebojno povezanost med izsledki ultrazvočne (UZ) preiskave TA in histopatološkimi podatki ter izražanjem izbranih miRNA na drugi strani. Metode: V prvem delu doktorskega dela smo z analizo izražanja miRNA pri bolnikih z GCA ocenili izražanje 28 miRNA v vzorcih TAB 30 bolnikov s histološko potrjeno boleznijo in izsledke primerjali z izražanjem miRNA v vzorcih TAB 16 bolnikov, ki niso imeli histološko potrjene bolezni, ter vzorcih TAB 22 oseb brez GCA. V drugem delu raziskave smo kvantitativno ocenili histološke spremembe v temporalnih arterijah bolnikov z GCA s histopatološkimi in imunohistokemičnimi tehnikami. V tretjem delu raziskave smo preverili, kako so najdbe UZ pregleda TA povezane s histolopatološkimi spremembami TA in z izražanjem miRNA. Rezultati: Z raziskavo izražanja miRNA smo ugotovili aberantno (več kot dvakratno) povišano ali znižano izražanje 28 miRNA. Aberantno izražene miRNA so vpete v patogenetske mehanizme, ki sodelujejo pri preoblikovanju arterij in uravnavanju odziva imunskega sistema. V raziskavi povezanosti histopatoloških lastnosti infiltrata in izražanjem miRNA smo ugotovili, da je vnetnocelični infiltrat sestavljen pretežno iz CD3+, CD4+, CD8+ limfocitov T, CD68+ makrofagov, ki jih je spremljalo povečano izražanje citoplazemskega jedrnega faktorja aktiviranih T celic 1 (NFATC). Pokazali smo, da bi spremenjeno izražanje izbranih miRNA lahko bilo povezano z infiltracijo žilne stene z limfociti in makrofagi ter aktivacijo limfocitov T. V raziskavi povezanosti histopatoloških, epigenetskih, kliničnih in slikovnih podatkov smo ugotovili močno povezanost prisotnosti halo znaka in debeline kompleksa intime-medije z intenzivnostjo vnetnega infiltrata, spremenjenim profilom izražanja miRNA in prisotnostjo klinične spremembe TA ter bolečine v žvečnih mišicah. Zaključki: Opredelitev profilov izražanja miRNA v steni temporalnih arterij bolnikov z GCA, ki potencialno sodelujejo pri uravnavanju vnetne infiltracije, imunskega odziva in fenotipa gladkih mišičnih celic, omogoča dodaten vpogled v zapleteno patogenezo GCA. Pokazali smo, da bi spremenjeno izražanje izbranih miRNA lahko bilo povezano z infiltracijo žilne stene z limfociti in makrofagi ter aktivacijo limfocitov T, ki so povečano izražali NFATC v TAB bolnikov z GCA. Naša raziskava omogoča vpogled v medsebojno povezanost parametrov UZ preiskave z ugotovljenimi histopatološkimi, epigenetskimi, kliničnimi in laboratorijskimi lastnostmi žilnih lezij pri bolnikih z GCA.Introduction: The main aim of this PhD thesis was to gain further insight into the pathogenetic mechanisms of giant cell arteritis (GCA) and to relate these to arterial ultrasound (nowadays the test of choice in the diagnosis of giant cell vasculitis). In the studies carried out as part of this PhD, we examined the expression of microRNAs (miRNAs) in temporal arteries (TA) of patients and their target genes. We phenotyped and quantified immune cells constituting the inflammatory infiltrate in temporal artery biopsies (TAB) from GCA patients and correlated the histopathological data obtained with miRNA expression. In addition, we evaluated the correlation between TA ultrasound (US) findings and, histopathological data and the expression of selected miRNAs on the other hand. Methods: In the first part of the study, we evaluated the expression of 28 miRNAs in TAB specimens from 30 patients with histologically confirmed disease and compared the results with the expression of miRNAs in TAB specimens from 16 patients without histologically confirmed disease and 22 subjects without GCA. In the second part of the study, we quantitatively assessed histological changes in temporal arteries of patients with GCA using histopathological and immunohistochemical techniques. In the third part of the study, we examined how findings on US examination of the TA correlated with histopathological changes of the TA and with miRNA expression. Results: The miRNA expression study revealed aberrant (more than twofold) elevated or decreased expression of 28 miRNAs. Aberrantly expressed miRNAs are involved in pathogenetic mechanisms involved in arterial remodelling and regulation of the immune response. In a study of the association between histopathological characteristics of the infiltrate and miRNA expression, we found that the inflammatory cell infiltrate consisted predominantly of CD3+, CD4+, CD8+ T lymphocytes, CD68+ macrophages, accompanied by increased expression of cytoplasmic nuclear factor of activated T cells 1 (NFATC). We showed that altered expression of selected miRNAs could be associated with infiltration of the vascular wall by lymphocytes and macrophages and activation of T lymphocytes. In a study of the association of histopathological, epigenetic, clinical and imaging data, we found a strong correlation between the presence of halo sign and the thickness of the intima-media complex with the intensity of the inflammatory infiltrate, the altered miRNA expression profile, the change in clinical appearance of TA and pain in the masticatory muscles. Conclusions: Identification of miRNA expression profiles in the temporal artery wall of GCA patients, potentially involved in the regulation of inflammatory infiltration, immune response and smooth muscle cell phenotype, provides new insights into the complex pathogenesis of GCA. We showed that altered expression of selected miRNAs could be associated with infiltration of the vascular wall by lymphocytes and macrophages and activation of T lymphocytes, which upregulated NFATC expression in the TAB of GCA patients. Our study provides insight into the correlation of ultrasound examination parameters with the histopathological, epigenetic, clinical and laboratory features of vascular lesions found in GCA patients

Evaluation of Arterial Histopathology and microRNA Expression That Underlie Ultrasonography Findings in Temporal Arteries of Patients with Giant Cell Arteritis

The aim of this study was to assess the interrelation between vascular ultrasonography (US) findings, histopathological data, and the expression of selected dysregulated microRNAs (miRNAs) in giant cell arteritis (GCA). The study included data on the clinical parameters, US measurements, and temporal artery biopsies (TABs) of 46 treatment-naïve patients diagnosed with GCA and 22 age-matched non-GCA patient controls. We performed a comprehensive comparative and correlation analysis along with generation of receiver operating characteristic (ROC) curves to ascertain the diagnostic performance of US examination parameters and selected miRNAs for GCA diagnosis. We showed significant differences in the US-measured intima–media thickness of the temporal arteries, the presence of a halo sign, and the presence of luminal stenosis between GCA-positive/TAB-positive, GCA-positive/TAB-negative, and non-GCA patients. Correlation analysis revealed significant associations between several histopathological parameters, US-measured intima–media thickness, and the halo sign. We found that the significant overexpression of miR-146b-5p, miR-155-5p, miR-511-5p, and miR-21-5p, and the under-expression of the miR-143/145 cluster, miR-30a-5p, and miR-125a-5p, coincides and is associated with the presence of a halo sign in patients with GCA. Notably, we determined a high diagnostic performance of miR-146b-5p, miR-21-3p, and miR-21-5p expression profiles in discriminating GCA patients from non-GCA controls, suggesting their potential utilization as putative biomarkers of GCA. Taken together, our study provides an insight into the US-based diagnostic evaluation of GCA by revealing the complex interrelation of clearly defined image findings with underlying vascular immunopathology and altered arterial tissue-specific miRNA profiles

Inflammatory cell composition and immune-related microRNA signature of temporal artery biopsies from patients with giant cell arteritis

Objectives: The aim of this study was to quantitatively assess distinct immune cell subsets comprising inflammatory infiltrate in temporal artery biopsies (TABs) from patients with giant cell arteritis (GCA), and to link the obtained histopathological data with expression profiles of immune-related microRNAs (miRNAs). Methods: The study included 68 formalin-fixed, paraffin-embedded TABs from treatment-naïve patients, including 30 histologically positive GCA and 16 negative GCA TABs, and 22 control non-GCA TABs. Quantitative assessment of histological parameters was performed using histopathological and immunohistochemical techniques. miRNA expression analysis was performed by quantitative real-time PCR. Results: Intense transmural mononuclear inflammatory infiltrates in TAB-positive GCA arteries were predominantly composed of CD3$^+$, CD4$^+$ and CD8$^+$ T lymphocytes, and CD68$^+$ macrophages, accompanied by a strong nuclear overexpression of the nuclear factor of activated T cells, cytoplasmic 1 (NFATC) in the lymphocyte infiltrate fraction. Furthermore, TAB-positive GCA arteries were characterized by significant overexpression of nine pro-inflammatory miRNAs (miR-132-3p/-142-3p/-142-5p/-155-5p/-210-3p/-212-3p/-326/-342-5p/-511-5p) and a significant under-expression of six regulatory immune-related miRNAs (miR-30a-5p/-30b-5p/-30c-5p/-30d-5p/-30e-5p/-124-3p), whose expression levels significantly associated with most evaluated histopathological parameters. Notably, we revealed miR-132-3p/-142-3p/-142-5p/-155-5p/-212-3p/-511-5p as major promoters of arterial inflammation and miR-30a-5p/-30c-5p/-30d-5p as putative regulators of NFATC signaling in TAB-positive GCA arteries. Conclusion: Overall, we demonstrated that an altered arterial tissue-specific pro-inflammatory miRNA signature favors enhanced T cell-driven inflammation and macrophage activity in TAB-positive GCA arteries. Moreover, dysregulation of several immune-related miRNAs seems to contribute crucially to GCA pathogenesis, through impairing their regulatory activity towards T cell-mediated immune responses driven by the calcineurin (CaN)/NFAT signaling pathway, indicating their therapeutic, diagnostic and prognostic potential

Data_Sheet_1_Milder outcomes of SARS-CoV-2 genetically confirmed reinfections compared to primary infections with the delta variant: A retrospective case-control study.pdf

BackgroundSARS-CoV-2 infection does not confer long immunity. However, studies suggest that prior infection is associated with lower risk of reinfection and milder outcomes of recurrent infections. The aims of this retrospective observational case-control study were to describe the clinical and molecular characteristics of genetically confirmed Delta reinfection cases and to assess the potential protective role of preceding infection on the severity of reinfection.MethodsWe used next generation sequencing (NGS) to explore if cases with two positive real time RT-PCR tests > 90 days apart were infected with a different SARS-CoV-2 variant. Cases with confirmed reinfection between August 1st and October 31st, 2021 (the Delta wave) in Slovenia were matched 1:4 by age, sex and timeframe (week of positive test) with individuals with primary infection. Sociodemographic and epidemiologic data, vaccination status, and data on hospitalization and outcome of infection were retrieved from several centralized and standardized national databases. Additional epidemiologic surveys were performed on a limited number of cases and controls.ResultsWe identified 628 cases of genetically confirmed reinfection during the study period and matched them with 2,512 control subjects with Delta primary infection. Primary infections in individuals with reinfection were mainly caused by B.1.258.17 (51.1%), followed by B.1.1.7 (15.1%) and reinfection was detected on average 271 days after primary infection (range 101–477 days). Our results show a substantially lower probability of hospitalization in cases with reinfection compared with controls (OR: 0.21, p = 0.017), but no significant difference was observed in intensive care unit admission and deaths. We observed a significantly lower proportion of vaccinated individuals among cases compared to controls (4.5% vs. 28.2%), suggesting that hybrid immunity leads to lower probability of reinfection. Detailed analysis of the temporal distribution of variants, responsible for reinfections, showed no significant differences in reinfection potential.ConclusionReinfection with the SARS-CoV-2 Delta variant resulted in fewer hospitalizations compared to the primary Delta infection, suggesting that primary infection may, to some extent, produce at least short lasting protective immunity. This study provides additional insight into the reinfection dynamics that may allow appropriate public health measures to be taken in subsequent waves of the COVID-19 pandemic.</p